The present invention relates to a method and apparatus for the calibration and quality control of a spectrophotometer and particularly an ELISA spectrophotometer used in a clinical laboratory.
Spectrophotometers are a well known tool in the analytical chemistry laboratory. One type of commercially available spectrophotometer is the ELISA spectrophotometer which typically comprises a plurality of light sources and detectors commonly arranged in a column of eight. The ELISA typically operates at one of two discrete wavelengths, such as 405 nm and 492 nm corresponding to yellow and red, respectively. The ELISA spectrophotometer can be used to analyze the photometric density produced by assay of biological materials. These assays are arranged as an assay plate having a number of rows corresponding to the number of channels of the ELISA spectrophotometer and a number of columns. Typically the ELISA spectrophotometer comprises an 8.times.12 matrix of 96 cells. The ELISA spectrophotometer now typically includes a microprocessor analyzing and recording the output of each channel for each assay well of a sample plate.
The ELISA spectrophotometer is a relatively recent addition to the analytical laboratory. An even more recent development has been the introduction of the ELISA spectrophotometer into the clinical laboratory. The immuno-chemical identification of exposure to hepatitis B virus, the Herpes virus, and the HIV virus, the "AIDS" virus, uses an ELISA spectrophotometer. The significance of photometric measurements made with an ELISA spectrophotometer now have implications that directly relate to the control of infectious epidemics. The measurement integrity of an ELISA spectrophotometer is therefore a matter of considerable concern to laboratory technicians, regulatory agencies and the general public.
The ELISA spectrophotometers used in clinical laboratories, however, are not equipped to insure proper calibration or quality control. Calibration is defined as the integrity of the normal operation of the instrument and relates to the spectrophotometer itself. Quality assurance is defined as the integrity of the results produced by a laboratory technician using a properly functioning spectrophotometer. At present, the calibration of an ELISA spectrophotometer is established once at the factory when manufactured. No provision is made for confirming calibration after the spectrophotometer leaves the factory. Quality assurance is left to each individual clinical laboratory and laboratory technician.
The calibration of an ELISA spectrophotometer can be compromised through both electronic and optical errors. Electrical errors arise from a variety of causes. An ELISA spectrophotometer employs filters of predetermined density and color. An electronic mechanism selects among the filters. A failure in the selection mechanism may result in the wrong filter being inserted. A laboratory technician would not notice the malfunction even if he could view the filter.
Alternately, the electronic memory that serves the microprocessor of the ELISA spectrophotometer may fail. Such a failure would most likely remain undetected using current calibration techniques. At present, an ELISA is calibrated by "blanking" the channels to establish a base line for zero optical density. A defective memory would likely read zero during "blanking". Nothing about the reading would necessarily indicate that the ELISA was defective. A memory defect used in the context of HIV screening would preclude the production of any positive test results. Individuals exposed to a virus would test free of infection whether or not such is true.
Yet another source of electronic error is the connection between the ELISA spectrophotometer and its microprocessor. The microprocessor of a personal computer often analyzes the output of the ELISA spectrophotometer and serves as the microprocessor for the ELISA. The transmission line between the spectrophotometer and personal computer normally uses a "hand-shake" protocol in which the photometer generates a check sum which is then exchanged with the computer. However, most programs used to analyze the output of an ELISA spectrophotometer do not analyze the check sum. Any transmission error thus goes unrecognized.
Optical errors can originate from a number of sources. For example, dust can obstruct a channel of an ELISA spectrophotometer and thus reduce its throughput efficiency. Alternately, the light source for a particular channel may become erratic and produce "jumps" in output or "burn hot" and produce a consistently high signal. This type of erratic output cannot be corrected using baseline subtraction.
Yet another source of potential optical error involves the deterioration of the filters of the ELISA spectrophotometer. This deterioration can take many forms such as, for example, the formation of cracks. Filter deterioration which is not necessarily noticed by the human eye can nevertheless give erroneous readings.
Optical errors can produce either false positives or false negatives depending on the test being run. The resulting misdiagnosis is traumatic to the patient involved and results in a substantial expenditure of time and resources to correct.
A second type of error in an ELISA spectrophotometer measurement is human error. A filter could be improperly inserted due to any number of reasons such as improper labeling or a defective selecting mechanism. A laboratory technician also could select the wrong filter for a given measurement. In either event, the error is not readily apparent using base line substraction because the values of the baseline measurements are substantially lower than those corresponding to a sample. Inserting the wrong filter causes all samples in a particular assay to appear "normal". The purpose of the assay is compromised and individuals are again diagnosed as being free of infectious diseases whether or not such is true.
The near total absence of calibration and quality assurance controls for ELISA spectrophotometers is uncharacteristic of the clinical laboratory. Stringent governmental regulation is more the norm than the exception. These regulations typically include frequently documented calibration tests of pipettes, scales, etc. Records must also be kept documenting preventative maintenance performed on the equipment as well as record that identify the equipment used to obtain the quality control and calibration measurements. For example, radiochemical procedures use a stable radioisotopes in combination with the counting equipment for daily quality assurance and calibration measurements. Records are maintained for review by the appropriate government regulatory agency. Likewise, test tube immunochemical procedures employ a series of sealed test tubes having dilutions of known color for use in a one channel photometer. The quality assurance measurements and calibrations are comparable to that required for radiochemical procedures.
The quality assurance and calibration confirmation procedures employed with a single channel photometer are not adequate for more complicated clinical procedures. For example, primitive "spot-check" calibration and baseline measurements are adequate for an ELISA spectrophotometer when used in an analytical laboratory. A skilled researcher could readily determine if his equipment or his procedure were defective since he would be highly familiar with the equipment and would have some idea of what result to expect. However, the clinical laboratory technician must analyze unknown samples without intuition. Errors are not apparent. Any errors on spectrophotometric measurement become matters of public health concern rather than strictly setbacks to research.
A need exists in the art for a method and apparatus for the calibration and quality assurance of ELISA and similar spectrophotometers that will work reliably and quickly in a clinical laboratory.